USPTO Patents Application 10561839

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization

International Bureau

(43) International Publication Date (10) International Publication Number

22 November 2001 (22,11.2001) pCT WO 01/88185 A2

(51) iDtemational Patent Classification^:

(21) International Application Number: PCT/GBO 1/021 22

(22) International Filing Date: 14 May 2001 (14.05.2001)
(25) Filing Language: English

C12Q 1/68 (81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX. MZ, NO, NZ, PL, FT, RO, RU. SD, SE, SG, SI, SK,
SL. TJ. TM, TR. TT. TZ. UA, UG, US. UZ. VN. YU. ZA,
ZW.

(26) Publication Language:

(30) Priority Data:
0011443.9

13 May 2000 (13.05.2000) GB

(71) Applicant (for all designated States except US): DNA
RESEARCH INNOVATIONS LIMITED [GB/GB];
940 Comforlh Drive, Sittingboume Research Centre.
Sittingboume, Kent ME9 8PX (GB).

(72) Inventor; and

(75) Inventor/Applicant (for US only): BAKER, Matthew,
John [GB/GB]; 481 Loose Road, Maidstone, Kent ME15

9UJ (GB).

English (84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC. NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG. CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).

Published:

— without international search report and to be republished
upon receipt of that report

For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette,

(74) Agents: KIDDLE> Simon, X et al.; Mewburn Ellis, York
House, 23 Kingsway, London, Greater London WC2B 6HP
(GB).

00

00
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(54) Title: SEPARATION DEVICE

(57) Abstract: A filter element for use in separation or purification of biomaterials such as nucleic acids fix>in solid contaminants
such as cell debris is described, having an end wall against which debris can collect and a side wall through which filtration can
occur, in the presence of debris layered against the end wall. The filter elements are preferably formed from a porous, rigid plastic
and are adapted to fit in a syringe, pipette or tube.

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Separa-bion Device
Field* of -the Invention

The present invention relates to separation devices and
5 in particular to filter elements ^ devices and methods for
separating solid contaminants from a liquid sample
containing nucleic acid.

Background of the Invention

10 When cells are lysed a crude mixture of soluble and

insoluble materials is obtained which often needs to be
purified for further analysis or purification. It is
often necessary to separate nucleic acid present in the
resulting mixture from solid contaminants and this

15 presents a difficult problem in the art, especially where
high throughput or automated sample processing are
required. In general, the preferred prior art method for
separating solid contaminants from nucleic acid
containing liquid samples is to use centrif ugation to

20 spin down the solid contaminants, leaving a liquid sample
containing the nucleic acid. However, while this is an
effective technique, it is a slow, labour intensive batch
process which is not readily amenable to automation and
also requires expensive equipment. Attempts to solve

25 this problem using conventional filters or membranes have
been unsuccessful as they need to be supported
(especially when wetted) and suffer from clogging, a lack
of robustness which adversely affects performance anci
working life for this type of purification.

30

SummarY Invention

Broadly, the present invention relates to filter elements
which can be incorporated in apparatus and used to
separate nucleic acid in liquid samples from solid

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contaminants. In particular, the present invention
relates to filter elements formed from' porous materials,
especially plastic material with rigid, porous structures
that can be formed in shapes other than the conventional
5 disk shaped filters. In preferred embodiments, the

filter elements of the present invention are formed with
an end wall against which solid contaminants tend to
collect, with filtration continuing to take place through
one or more unblocked side walls, e.g. in a lateral
10 direction as compared to the flow of the liquid sample

against the end wall. In particular, the working life of
the filter elements and their adaptability makes the
present invention suitable for a range of different
situations and can be used in automated systems.

15

Accordingly, in a first aspect, the present invention
provides a filter element formed from a material having a
rigid porous structure with a pore size between about 10
and about 200 microns, the filter element having an end

20 wall and one or more side walls extending out of the
plane of the end wall, so that when a liquid sample
comprising nucleic acid and solid contaminants is
introduced into the element, the liquid containing the
nucleic acid filters through the side and/or end walls,

25 while the solid contaminants are retained.

In one embodiment, the filter element is tubular and has
a closed end so that when a sample comprising liquid and
solid material contacts the filter element, e.g. when it

30 is drawn into a pipette tip or syringe in which the

filter is retained, the liquid filters through the side
walls and out of the open end of the tube, while the
solid material is initially builds up and is retained on
the end wall. Thus, in this embodiment, the tubular part

35 of the element forms the side walls, while the outside of

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closed end of the tube provides the end wall. In use, as
solid material builds up in the closed end of the filter
element, the liquid containing the nucleic acid can pass
through the side walls, allowing filtration to continue
5 and increasing the working life of the filter. In this
embodiment, the tube preferably has a uniform circular
cross-section. However, other cross-sections will be
apparent to those skilled in the art and may be employed
to adapt the filter element to fit in apparatus of
10 differing geometries, at locations in the apparatus where
a filtration function is required. It would also be
possible to include a taper in the filter element, i.e.
so that the cross-section varied along its length.

15 In an alternative embodiment, the filter element is in

the form of a plug for spanning an aperture in a piece of
apparatus, such as a tube or pipette tip, the plug having
an end wall adapted to retain the filter element in the
aperture and a side wall protruding from the end wall.

20 In one preferred embodiment, the plug is approximately T-
shaped in cross section, and the side wall protruding
from the end wall has a circular cross section. In use,
the liquid sample is introduced around the protruding
side wall and can filter through the end wall' and the

25 protruding side wall. As in the embodiment above, even

if the end wall becomes blocked with solid debris, liquid
can still pass for some time through the protruding
portion as it is raised above the plane of the end wall.

30 In embodiments where the filter element is adapted to fit
inside a pipette tip, a syringe or small tube (e.g. a PGR
or centrifuge tube) , conveniently, it has a diameter of
between about 5 and 15mm and a length of between about 10
and 2 0 mm. In the first type of tube filter element,

35 preferably the inner diameter of the tube is between

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about 3 and 5 mm. In the second type of filter element
having a protruding side wall, preferably this has a
diameter of between about 3 and 6 mm,

5 In the present invention, the end wall and side walls are
defined in relation to the flow of the liquid sample
through the apparatus containing the filter element. In
preferred embodiments, the side wall(s) of the filter
element away from the plane of the end wall so that even
10 if the' end wall becomes blocked by layers of solid

contaminants or debris building up on it, filtration
through the parts of the side wall above the level of the
blocking debris is possible.

15 The filter elements of the present invention therefore
provide a solution to the unsolved problem in the prior
art of filtering solid debris from liquid samples
containing nucleic acid. In preferred configurations,
the filter elements are capable of quickly filtering even

20 large volume samples. In comparison to conventional

cellulose or glass fibre paper type filters, the filter
elements of the invention typically retain less of the
liquid sample in the filter, an important advantage in
this context as nucleic acid containing liquid sample are

25 often low voliame.

The present invention can further be readily adapted for
automatic processing in an 8 x 12 format and a
standardised pitch where an increased diameter will

30 prevent or hinder multi-channel filtration. The geometry
of the filter elements of the invention, which are
generally longer than they are wide, works well in these
situations, especially when assisted by suction. In this
case, typically the length of the filter elements is

35 greater than the width and more preferably at least 1.5

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times the width, and still more preferably 2 times the
width. The width of the filter element is measured
parallel to the plane of the end wall, with the length of
the filter element measured parallel to the plane of the
5 side wall (s) .

A preferred material for making the filter elements is a
porous plastic material such as polypropylene, high
density polyethylene (HDPE) , polytetraf luoroethane

10 (PTFE), nylon or polyether sulphone. These materials are
readily available as sintered plastics and can be formed
into the rigid filter elements having the shapes
described above. Alternatively, sintered glass could be
employed, or an alternative silica, glass or ceramic

15 material •

Preferably, the filter element has a pore size between
about 0.01 microns and about 500 microns, more preferably
between about 10 microns and about 200 microns, and more
20 preferably between about 20 and about 50 microns. For
the filtration of nucleic acid samples,, the present
inventors have found a pore size between about 10 and
about 30 microns to be optimal.

25 In a further aspect, the present invention provides an
apparatus comprising a filter element as described
herein.

In a further aspect, the present invention provides a kit
30 comprising a plurality of the filter elements. and

optionally apparatus into which the filter elements are
adapted to fit.

In a further aspect, the present invention provides the
35 use of a filter element as described herein for filtering

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solid contaminants from a liquid sample containing
nucleic acid.

In a further aspect, the present invention provides a
5 method of filtering a liquid sample comprising nucleic
acid and one or more solid contaminants / the method
comprising passing the sample through a filter element as
described herein so that the liquid containing the
nucleic acid passes through the filter element and the
10 solid contaminants are retained by the filter element.

In a preferred embodiment, the method includes the
initial step of lysing a cell culture to provide a sample
and precipitating proteins present in the sample, e.g.
15 with sodium doceyl sulphate (SDS) . This commonly used
method to prepare samples results in a large amount of
solid material that cannot be filtered efficiently using
prior art techniques.

20 According to the invention there is provided a filter

element which comprises a sintered material adapted to be
moulded to produce a rigid porous structure and the
invention also provides a filter which incorporates such
a filter element.

25

Preferably the filter eleitlent provides a large surface
area, e.g. it is in the form of a hollow plug with the
length longer than the width for example with the ratio
of length to width of at least 1.5:1, and more preferably
30 at least 2.0:1.0.

An example of a separation device that incorporates the
element of the invention uses the element in a multi-
channel array, e.g. an 8 x 12 array.

35

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Preferably the device is comprised of a rigid, mouldable,
self-supporting porous plug, composed of sintered porous
plastic or glass, that can be attached to a pumping or
sucking system- The porous plug may be modified
5 chemically or by adsorption of ligands to specifically
capture target compounds or remove unwanted materials.

The device may be any shape with a cross sectional area
to maximise surface area. Preferably, the devices are

10 longer than they are wide to maximise surface area but

maintaining a low diameter for insertion into tubes. The
rigid wicks or hollow plugs may be nested inside each
other to create a sequence of filters or the hollow plug
may contain further particles or microfibers to filter

15 out fine material. Alternatively, a large number of
smaller plugs may be used in parallel to provide even
larger surface areas .

The device may be combined with chromatographic or
20 affinity purification using standard solid-phases, e.g.
ion-exchange. Protein A, antibodies, Streptavidin, etc*

The device is particularly useful for the filtration or
purification of biomolecules and cells and especially for
25 separating nucleic acids from liquid mixtures.

In use the liquid to be purified, or from which solid
material is to be separated, is drawn up through the
filter element into a reservoir or. other receptacle.

30

The invention is particularly useful to remove cell
debris from lysed cells.

One embodiment of the invention allows a crude extract of
35 insoluble or soluble materials to be sucked up into a

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reservoir from a range of laboratory test tubes such as
PGR tubes, micro-tit re plates, centrifuge tubes and any
standard container from a few microlit'res to litre
voliames. Once the fluids have been drawn up through the
5 device then further processing or purification is
possible.

The shape and design of the device is flexible and may be
formed by moulding the porous material into any shape or
10 structure.

It is a feature of the device that it can maximise flow
rates, prevent clogging or blockages and presents a
larger than normal surface area parallel to the fluid in
15 both directions while maintaining a narrow diameter for
multi-channel fluid handling systems.

The filter element is self supporting and rigid, not
requiring other supporting casing- or moulds for it to
20 work. Therefore it can be placed over the outside. of a
dispensing or aspirating system and removed to discard
the filter element or to process the material captured by
the filter element.

25 The filter element can be incorporated internally in a
pipette or can be attached to the end of a pipette so
that liquid can be sucked up through the filter element
into the pipette .

30 Several designs of plug have been tested for efficient
separation of contaminants, using cellulose or glass
microfiber membranes. Another variation for microbial
purifications is that the cells or debris can be
concentrated or removed by using specific ligands such as

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antibodies, polymixins, lectins, enzymes, boronic acid or
other affinity materials.

5

The device is especially suitable for biological samples
5 from medical research to food and agriculture where
insoluble materials need to be removed before
purification of the target analyte.

Embodiments of the present invention will now be
10 described in more detail by way of example and not

limitation with reference to the accompanying drawings.

Brief Description of the Drawings

Figures 1 and 2 shows embodiments of the invention and in
15 place in different apparatus where a filtration function
is needed.

Figures 3a and 3b show an example of a closed tubular
embodiment of the invention..

20

Figure 4 shows an example of a filter element with a
protruding side wall, in place in a centrifuge tube.

Figure 5 shows a perspective view of the filter element
25 of Figure 4.

Figure 6 shows the filter element of Figure 4 or Figure 5
in place in a pipette tip..

30 Detailed Descripliion .

Figure la shows a syringe 1 sucking up a plasmid
preparation 3 through a hollow porous plastic plug 2 with
the bottom end 4 closed. The debris remain on the"
outside of the plug allowing the DNA to travel through
35 into the syringe barrel. The plug 2 avoids immediate

9

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blockage by presenting a large surface area and the
external housing of the cartridge allows the material to
travel up the plug without forcing the particulate
material into the pores. The plug 2 only blocks when the
5 liquid has travelled all the way to the top of the

housing- The filtration device may then be removed and
the liquid transferred into a new tube. The use of this
embodiment is described in Examples 1, 2 and 3.

10 The above system allows fully automated extraction of

plasmid DNA from crude bacterial lysates. The reservoir
above the filter holds the clarified fluid for
purification on affinity media of some type.

15 The device has many applications whether in a manual or
automated operation and even with larger volxames a
standard eight by twelve array of tubes can be processed
without a pitch change in a multi-channel instriament.

20 Figure lb shows a plug 2 in a pipette tip 5 so that, if
the plasmid lysate is clarified, then the plug may be
modified to capture DNA directly.

Figure Ic shows a modification of the Figure la device
25 without the external housing surrounding the hollow plug
2.

Figure Id shows a porous plug or hollow plug 2 fitted on
externally to allow easy removal while maintaining the
30 fluid* in the pipette tip 5-

Figure le shows a porous plug fitted onto a solid pin or
moulding that can be dipped into a tube to capture
biomolecules . This could be extended to an 8 x 12
35 microtitre foinoaat or. PGR tube array.

10

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Figure If shows, embodiments of the plugs 2 of the
invention which are shaped to increase the surface area
in standard pipette tips, with end walls 7 and side walls
5 6 marked.

Figure Ig shows the outline of a hollow porous plug made
from sintered plastic or glass, showing the side walls 6
and end wall 7 of the plug 2. The device is rigid enough
10 to support itself and the open end is fitted onto the
sucking and pumping system. This design maximises
surface area vertically and reduces the pitch between
adjacent devices,, e.g. in a multi-channel system.

15 In Figure 2a there is a pump 5 that can generate

continuous liquid flow through the device incorporating
plug 6 so that the liquid may be re-circulated if
required.

20 Figure 2b shows how the device may be used with

centrifugation tubes to increase the surface area
compared to a flat disc where 7 is the liquid and 8 is
the filter element.

25 Figure 3 shows an embodiment of the invention which uses
a filter element 8 having the foirm of a tubular plug 10
having a closed end 12 and an open end 14, with arrows
showing the flow of a liquid sample through the filter
element. The external surface of the closed end 12

30 provides an end wall 18 and the curved surface of the
tubular part of the plug defines a side wall 20. The
filter element 8 is retained in a syringe, pipette or
other tube 16 with the closed end 12 of the plug directed
towards the flow of the sample into the tube 16. When a

35 sample encounters the filter element 8, solid

11

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contaminants^ such as cell debris^ will tend to be
retained on the end wall 18, while liquid containing
nucleic acid and other soluble components of the sample
can pass through the side wall 20 into the hollow core of
5 the tube and out of the open end 14 for further

purification or analysis, the hollow core helping the
efficiency of filtration by reducing the transmembrane
pressure experienced by the sample across the filter
element. The tendency of the end wall of the device to

10 capture debris and the high surface area that results

from using a porous plastic material to form the filter
element 8 means that the rapid clogging observed with
prior art filtration techniques is avoided^ and that
instead layers of solid debris tend to build up on the

15 end wall of the device.

Figure 4 shows an alternative form of filter element 8,
in this case designed to fit across the opening of a
centrifuge or PGR tube 22. The filter element has an end

20 wall 18 which spans the opening 24 of the tube 22 and a
generally cylindrical central portion 26 having a side
wall 20 which protrudes towards the direction of sample
flow. In use, a sample introduced into the open space at
the top of the tube 22 can filter, through the end wall 18

25 and side wall 20, with debris again tending over time to
collect against the end wall, leaving the liquid free to
filter through the side wall 20 as the protruding central
portion stands clear of the build up the solid. debris.
Figure 5 shows a perspective view of the filter element

30 8, while Figure 6 shows the filter element in place in a
pipette tip 28. As in Figure 3, the arrows indicate the
direction of liquid flow through the filter element.

Exainple 1

35 Extract:xon o£ nucleic add from bacterial lysates

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This example demonstrates the filtration of bacterial
lysates and the purification of plasmid DNA. An
overnight culture of E. Coll possessing a plasmid was
lysed using a modified alkaline lysis method and the cell
5 debris were removed by sucking the fluid up through a
rigid 20 micron porous sintered plastic plug using
embodiment shown in Figure la. The debris was retained
by the filter allowing the plasmid DNA to travel into the
reservoir in this case a syringe barrel or pipette tip.

10 The plasmid DNA was captured on the modified plug and

washed free of contaminants with water before recovery in
a small volume of Tris.HCl pH8.5. The plug was removed
and the fluid allowed to be pumped down through another
plug covalently modified with polyhistidine according to

15 patent application WO 99/29703 (DNA Research Instruments
Ltd) .

Example 2

Extraction of nucleic acid from natural source material

2 grams of cabbage leaves were homogenised in warm sodium
dodecyl sulphate (SDS) to release the nucleic acids.
Following potassium acetate/potassium chloride
precipitation^ the fluid was sucked up a twenty micron
plug to remove the insoluble material and the DNA
extracted using a polyhistidine affinity membrane
combined in the device.

Exanqple 3

Extraction of nucleic acid from white blood cells.

20

25

r

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Affinity capture of analytes such as nucleic acids,,
proteins, cells , organelles and other compounds were
performed using this device. The capture or removal of
white blood cells from whole anti-coagulated blood can be
5 performed by mixing the blood with ammonium bicarbonate
buffers containing high levels of- non ionic detergents
such as 1% (v/v) Tween 20.

The blood is sucked through a hollow plug allowing the
10 cells to bind and the contaminants washed off using the
same buffer. The cells may then be processed for
collection of DNA, RNA or analysed by a known method.
This system can be used in combination with collection of
blood samples directly from the donor either using a
15 needle and syringe or a vacuum tube to suck the blood

through the porous material. The porous material may be
used to store the captured substance or transferred to
another storage tube without having to* release the
captured substance.

20

Example 4

Extraci:lon o£ plasmld DNA from cull^ure

An overnight culture of E.Coli/PUC19 was prepared and
25ml centrifuged to pellet the cells. The cell pellet

25 was resuspended in 2ml of lOmM Tris HCl containing Rnase
A and mixed with a further 2ml of 0.2M NaOH with 1%SDS to
lyse the cells and release the plasmid DNA. The cellular
debris and SDS was then precipitated with 2ml of 3M
potassixim Acetate pH4 and left to stand for 5 minutes.

30 The liquid was separated from the precipitate by a filter-
element of the type depicted in Figure 3^ using a 25
micron pore plastic hollow plug inside a 3ml cartridge
about 4cm long and 1cm in diameter. The cartridge tip
was dipped into the mixture and the liquid sucked up *

35 through the filter into a syringe barrel. The

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precipitate remained on the outside of the porous plug
producing a clear liquid in the syringe barrel in about 1
minute. The total yield of liquid was 5.5ml, over 90%
recovery from the starting material . The filtered liquid
5 was then processed to obtain pure plasmid using magnetic
beads derivatised with Bis-Tris or by alcohol
precipitation.

The filter plug was then regenerated by pumping water
10 back through until all the precipitated was washed away.
This can then be used for repeat experiments or
continuous flow operation.

The same experiment was repeated except the cartridge was
15 inverted and the precipitated mixture was piimped from the
syringe barrel down through the plug. The precipitate
collected at the base of the plug leaving the majority of
the filter unclogged to allow easy flow of liquid. In
this case, recovery of liquid was even better at about
20 95% yield.

The device was used as a pre-filter on the same volume of
plasmid preparation to allow filtering down to 1 micron
or 0.45 micron. By incorporating an additional filter
25 after the plug, the device allowed filtration to 1 micron
or less with 80% recovery of fluid and a 5 minute
filtration time.

Exainple 5

30 Coniparxson with conventional fll'bra'bion

Instead of using the. filter element described above, a
standard 25 micron pore frit made of porous plastic sheet
with a diameter of 25mm was inserted into a 30ml syringe
barrel with spacing collars to hold it in place and
35 expose the surface to the liquid.

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The precipitated mixture from the plasmid preparation was
either sucked up through the frit or pushed through. In
both cases, only 50% of the fluid was recovered due to
5 almost immediate clogging of the membrane. In an attempt
to prevent clogging, stacks of filter paper were placed
in front of the 25mm frit, but the performance in terms
of yield of liquid and flow rates could not be improved.

10 Thus, if standard 25inm glass fibre or paper pre-f liters
are used, clogging occurs very quickly and recovery of
liquid is slow. In many cases, this means that it is
impractical to use filtration to remove solid
contaminants from liquid samples containing nucleic acid.

15

Filtration in mxcrotubes using cehtrif ugation vacuum
manifolds

A conventional frit or filter from a 1.5ml centrifuge
filter tube was replaced with a porous 25 micron plug

20 inverted to increase the surface area and prevent

clogging. A 5ml culture was precipitated as described
above reducing the original volume to about 1ml ready for
filtration. The mixture was tipped into the tube with
the plug and either placed on a vacuum manifold or

25 centrifuged for 3 minutes. The fluid was easily

collected with no signs of clogging and 90% of liquid was
recovered.

With the original filter material in place, clogging with
30 this sample volume occurred immediately and only about
50% of the original was recovered.

, Fxl-bralixon using plpe-bte tips

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A standard 1ml pipette tip was used to filter a 5ml
plasmid preparation by inserting a 25 micron plug into
the tip. The mixture could either be sucked up or pumped
through within 1 minute with 80% recovery of liquid.
5 This was then repeated using a multi-channel pipettor for
filtering 8 samples simultaneously.

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Claims :

1. A filter element formed from a material having a
rigid» porous structure with a pore size between about 10
and about 200 microns^ the filter element having an end

5 wall and one or more side walls extending out of the
plane of the end wall, so that when a liquid sample
comprising nucleic acid and solid contaminants is
introduced into the element , the liquid containing the
nucleic acid filters through the side and/or end walls,
10 while the solid contaminants are retained.

2. The filter element of claim 1.^ wherein the filter
element is a close ended tube with the side wall defined
by a curved wall of the tube and end wall defined by the

15 outside of closed end of the tube.

3. The filter element of claim 1^ wherein the filter
element is in the form of a plug for spanning an aperture
in a piece of apparatus, and having an end wall from

20 which a side wall protrudes.

4 . The filter element of any one of claims 1 to 3 which
is adapted to fit into a pipette tip, a syringe or a PGR
or centrifuge tube.

25

5. The filter element of any one of the preceding
claims, wherein the material is a plastic.

6. The filter element of any one of the preceding

30 claims wherein the plastic is polypropylene, high density
polyethylene (HOPE), polytetraf luoroethane (PTFE) , nylon
or polyether sulphone.

7. The filter element of claim 5 or claim 6, wherein
35 the plastic is a sintered plastic.

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8. The filter element of any one of the preceding
claims, wherein the pore size is between about 20 and
about 50 microns;

5

9. The filter element of any one of the preceding
claims, wherein the length of filter element is greater
than its width.

10 10. The filter element of claim 9, wherein the ratio of
length to width is at least 1.5:1.

11. Apparatus comprising a filter element of any one of
the preceding claims.

15

12. The apparatus of claim 11 which is a pipette tip, a
multipipettor, a syringe, or a PGR or centrif ugation
tube,

20 13. A kit comprising a plurality of the filter elements
of any one of claims 1 to 10 and optionally apparatus
into which the filter elements are adapted to fit.

14. Use of a filter element of any one of claims 1 to 10
25 for filtering solid contaminants from a liquid sample

containing nucleic acid.

15. A. method of filtering a liquid .sMiple comprising
nucleic acid and one or more solid contaminants, the

30 method comprising passing the sample through a filter

element of any one of claims 1 to 10 so that the liquid
containing the nucleic acid passes through the filter
element and the solid contaminants are retained by the
filter element.

35

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16. The method of claim 15, wherein the liquid sample is
a cell culture and the method includes the initial step
of ly^ing a cell culture and precipitating proteins
present in the sample-

17. The method of claim 15 or claim 16^ comprising the
step of sucking the liquid sample- through the filter
element .

20

wo 01/88185

PCT/GBOl/02122

1/3

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SUBSTITUTE SHEET (RULE 26)

wo 01/88185

2/3

PCT/GBOl/02122

Fig.2a.

Fig.2b.

SUBSTITUTE SHEET (RULE 26)

wo 01/88185

PCT/GBOl/02122

(12) INTERWATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization

International Bureau

(43) International Publication Date
22 November 2001 (22.11.2001)

PCT

IllllllllillillllillllW^

(10) International Publication Number

wo 01/88185 A3

(51) International Patent Classification'': C12Q 1/68,

C12N 15/10, BOID 39/02

(21) International Application Number: PCT/GBO 1/02 122

(22) International Filing Date: 14 May 2001 (14.05.2001)

(25) Filing Language: English

(26) Publication Language: English

(30) Priority Data:
0011443.9

13 May 2000 (13.05.2000) GB

(71) Applicant (for all designated States except US): DNA
RESEARCH INNOVATIONS LIMITED [GB/GB];
940 Cornforth Drive, Sittingboume Research Centre,
Sittingboume, Kent ME9 8PX (GB).

(72) Inventor; and

(75) Inventor/Applicant (for US only): BAKER, Matthew,
John [GB/GB]; 481 Loose Road, Maidstone, Kent ME15

9UJ (GB).

(74) Agents: KIDDLE, Simon, X et al.; Mewbum Ellis, York
House, 23 Kings way, London, Greater London WC2B 6HP
(GB).

(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO. CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, H, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, NO. NZ, PL, PT, RO. RU, SD, SE, SG, SI, SK,
SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA,
ZW.

(84) Designated States (regional): ARJPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG, CI. CM, GA. GN, GW, ML, MR, NE, SN, TD. TG).

Published:

— with international search report

(88) Date of publication of the international search report:

4 April 2002

For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette,

00

oo

00

(54) Title: A FDLHER ELEMENT FOR USE IN SEPARATION OF BIOMATERLU-S FROM SOLID CONTAMINANTS

(57) Abstract: A filter element for. use in separation or purification of biomaterials such as nucleic acids from solid contaminants
such as cell debris is described, having an end wall against which debris can collect and a side wall through which filtration can
occur, in the presence of debris layered against the end wall. The filter elements are preferably formed from a porous, rigid plastic
and are adapted to fit in a syringe, pipette or tube.

INTERNATIONAL SEARCH REPORT

Inter 'onat Application No

PCi/GB 01/02122

A. CLASSIFICATION OF SUBJECT MATTER

IPC 7 C12Q1/68 C12N15/10 B01D39/02

Accordmq to International Patent Classittcation <IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (ciassiticalion system followed by classification symbols)

IPC 7 C12Q C12N BOID C12M

Documentation searched other than minimum documentation to ttie extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and. where practical, search terms used)

WPI Data, PAJ, EPO-Internal , BIOSIS. MEDLINE, CHEM ABS Data, EMBASE,
BIOTECHNOLOGY ABS, SCISEARCH

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category "

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

X

WO 97 26540 A (KEMPE TOMAS ;BARRSKOGEN INC

1-17

(US)) 24 July 1997 (1997-07-24)

page 8, line 16 -page 11, line 27

page 12, line 1 -page 14, line 24; claims

1-14; figure 1

page 14, line 22 - line 24

page 16, line 8 - line 21

page 13, line 19 - line 24

page 16, line 9 -page 17, line 2

abstract

X

WO 00 09680 A (CHIRON CORP)

1-17

24 February 2000 (2000-02-24)

page 2, paragraph 6

page 6, paragraph 4; figure 1

page 7, paragraph 2; claims 1-14

page 4, paragraph 4 -page 7, paragraph 1

-/~

Further documents are listed in the continuation of box C.

Patent family members are listed In annex.

** Special categories of died documents :

*A' document defining the general state of the art which is not

considered to be of particular relevance
*E' earlier document but published on or after the International

tHing date

*L* document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)

•O* document referring to an oral disclosure, use. exhibition or
other means

•P' document published prior to the international filing date but
later than the priority date claimed

•T" later document published after ttie international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention

'X* document of particular relevance: the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document s taken alone

•Y' document of particular relevance; the claimed invention

cannot be considered to involve an inventive step when the
document Is combined with one or nrrare other such docu-
ments, such combination being obvious to a person skilled
in the art.

'&* document member of the same patent family

Date of the actual completion of the international search

6 December 2001

Date of nrtaiting ot the internationat search report

18/12/2001

Name and mailing address of the ISA

European Patent Office. P.B. 5818 Patentlaan 2
NL - 2280 HV Rijswiik
Tel (+31-70) 340-2040, Tx. 31 651 epo nl.
Fax: (+31-70) 340-3016

Authorized officer

Tllkorn, A-C

Form PCT/1SA/210 (second sheet) (July 1992)

page 1 of 2

INTERNATIONAL SEARCH REPORT

Inter onal Application No

PCl/GB 01/02122

C.(Conttnuation) DOCUMENTS CONSIDERED TO BE RELEVANT

Category Citation ol document, with indication.where appropriate, of the relevant passages

Relevant to claim No.

wo 96 41810 A (DON ROBERT HUGH ; EVANS
TIMOTHY MARTIN (AU); PROGEN IND LIMITED
(AU) 27 December 1996 (1996-12-27)
page 2, line 21 -page 5, line 31; claims
1-62; figure 1

WO 00 25922 A (THE PERKIN-ELMER
CORPORATION) 11 May 2000 (2000-05-11)
abstract

page 23, line 28 -page 24, line 16;
figures 3-6

US 5 824 224 A (FUJISHIRO MASATOSHI ET
AL) 20 October 1998 (1998-10-20)
abstract

column 7, line 2 -column 17; claims 1-3

WO 96 08500 A (QIAGEN GMBH ;C0LPAN METIN
(DE)) 21 March 1996 (1996-03-21)
abstract; claims 1-14; figure 1

Fonn PCT/ISAei 0 (continualion ol aacond sheet) (July 1 9S2)

1-17

1-17

1-17

1-17

page 2 of 2

INTERNATIONAL SEARCH REPORT

•iformation on patent family members

Inter onat Application No

PCl/GB 01/02122

Patent document

Publication

Patent family

Publication

cited in search report

date

member(s)

date

WO 9726540

A

24-07-1997

Uo

CQQVQOQ A

Q79Ac;4n Al

y/cOO*»U Mi

WO 0009680

A

24-02-2000

us

6268492 Bl

31-07-2001

EP

1104458 Al

06-06-2001

UUU7Q0U rti

24-02-2000

WO 9641810

A

27-12-1996

Ail

Oyo/Ul

AU

5888096 A

09-01-1997

WO

9641810 Al

27-12-1996

PA

9991ftQfi Al
^^^OO^D Ml

?7-1 P-1996

tr

UojUoOH Mi

JP

11507544 T

06-07-1999

us

5989431 A

23-11-1999

MO 0025922

A

11-05-2000

CO'X^Q A
DibysJOo A

1 9-1 9-9nnn

i^ i^ ^UUU

AU

1 c 1 nn A

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20-10-1998

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WO 9608500

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21-03-1996

DE

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WU

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EP

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27-11-1996

EP

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28-05-1997

EP

0743949 Al

27-11-1996

EP

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02-07-1997

JP

9508406 T

26-08-1997

JP

9508283 T

26-08-1997

JP

9508407 T

26-08-1997

US

6297371 Bl

02-10-2001

US

5747663 A

05-05-1998

Foim PCT/ISA/210 (patent family emex) (July 1992)

page 1 of 2

INTERNATIONAL SEARCH REPORT

.nformation on patent family memt>ers

Inte iona) Application No

PCr/GB 01/02122

Patent document

cited in seaicti report

Publication
date

Patent family
member(s)

Publication
date

WO 9608500

US
US

5792651 A
6274371 Bl

11-08-1998
14-08-2001

Form PCT/ISA/210 (patent lamily amex) (July 1992)

page 2 of 2